Searching for the structural determinants of the substrate specificity and catalytic efficiency of the Ferredoxin-NADP(H) reductase

CECCARELLI, EDUARDO A; ARAKAKI, ADRIÁN K; CATALANO DUPUY, DANIELA L; RIAL, DANIELA V; MUSUMECI, MATÍAS; PALADINI, DARÍO H

Angra dos Reis, Brasil.

Congreso; 1st Latin American Protein Society Meeting.; 2004

Latin American Protein Society

Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that participate in a wide range of redox metabolic pathways in an ample variety of organisms. Several structural and functional aspects of these enzymes remain yet to be explained. In that context, chloroplast FNR has a 32,000- fold preference for NADP(H) over NAD(H), displaying turnover numbers that are 20- to 100-fold higher than FNRs from bacteria, albeit they share extensive structural and conformational identity. FNRs consist of two domains; one involved in the binding of the prosthetic group FAD and the other responsible for binding of NADP+. Noteworthy, two tyrosine residues lie close to each side of the isoalloxazine. The highly conserved Y308 in pea FNR is stacked near parallel to the re-face of the flavin and should be displaced by the nicotinamide ring of NADP(H) for productive binding to the enzyme. The other side of the flavin is facing Y89, which participates in a network of interactions with other amino acids and with the FAD itself. We have investigated the function of these two tyrosines performing ab initio molecular orbital calculations and site directed mutagenesis. Our results indicate that the position of Y89 is mainly governed by the energetic minimum of the pairwise interaction between the phenol ring and the flavin, displaying geometries that correspond to the more negative free energy theoretical value. By the contrary, the Y308 is constrained against the isoalloxazine by amino acids C266 and L268, which are facing the other side of this tyrosine, forcing the Y308 to adopt a more planar orientation with respect to the flavin. A set of aromatic and non aromatic mutants of both amino acids were obtained. Analyses of these FNR mutants indicate that aromaticity on residue 89 is essential for FAD binding and proper folding of the protein and, that hydrogen bonding through the Y89 hydroxyl group may be responsible for the correct positioning of FAD and the substrate NADP+. On the other hand, our results indicate that the Y308 plays a key role in modulating pyridine nucleotide discrimination and enhancing the enzyme efficiency. Simple and double mutants of amino acids C266 and L268 allow us to suggest that these amino acids participate in the fine-tuning of the enzyme eciency, modulating the interaction of the Y308 with the isoaloxazine.